There was a linear relationship of [alpha]-amylase activity
from 2 to 12 hours incubation in both freeze-dried and fresh
'd'Anjou' pear fruit tissue. Activity was greater, however,
in fresh as compared with freeze-dried tissues. [alpha]-Amylase
activity from both types of samples was not detected until
60 minutes incubation. After 90 minutes, activity increased
significantly in both freeze-dried and fresh tissue
extracts.
Three different buffers (acetate, Tris-HCl and
imidazole-HCl) were used at varying pH levels (from 4.60 to
8.23) to ascertain the optimum assay system. Highest
specific activity was recorded with an acetate buffer at pH
5.64. The Km value in this system was 1.43 mg.ml⁻¹.
Specific activity of [alpha]-amylase increased as Ca
concentration in the reaction mixture increased from 1 to 15
mM CaCl₂. No changes in specific activity were found as the
Ca concentration increased from 15 to 25 mM CaCl₂.
Alpha-amylase from 'd'Anjou' pear fruit was purified
5.68 fold using ammonium sulfate fractionation and a
desalting column (Bio-Gel p-6).
Activity of [alpha]-amylase and protein, calcium and starch
concentration were measured in preharvest calcium-treated
normal and corkspotted 'd'Anjou' pear fruit at commercial
harvest maturity. Activity and specific activity of [alpha]-amylase
extracted from corkspotted fruit were higher as
compared to [alpha]-amylase extracted from normal tissue. Protein
concentration was greater in corkspotted fruit. Starch
levels were less in corkspotted than in normal fruit as
evidenced either by an iodine stain visual technique or by
quantitative analysis of hot-water soluble starch. No
interaction were found with any measured parameter between
calcium treatment and fruit condition. Activity of [alpha]-amylase
extracted from calcium-treated pears was greater
than that extracted from normal pears. Corkspotted fruit
had less calcium as compared to normal fruit.